Autophagy is important in cancer development, progression and response to therapy. It is widely thought that these activities ultimately rely on autophagy's well-known ability to modulate tumor cell death. Although we are already trying to target autophagy in >40 clinical trials and are inadvertently manipulating autophagy both positively and negatively in thousands of other trials and with standard cancer therapy, a central knowledge gap hampers our efforts to maximally benefit from these efforts. This knowledge gap is that we still have very little mechanistic understanding of how autophagy controls tumor cell apoptosis. This grant is a renewal of a project focused on the core question- how does autophagy regulate tumor cell apoptosis and what does this mean for tumor treatment? The grant builds on three novel discoveries that were made in the previous funding period. First, we found that autophagy variation in a cell population determines which tumor cells live or die in response to a future apoptotic stimulus. Second, we uncovered a specific mechanism by which autophagy allows tumor cells to recover from and avoid apoptosis and explains how autophagy inhibition potentiates canonical apoptosis. Third, we discovered that autophagy inhibition in dying tumor cells promotes growth of therapy-resistant sub-clones in the population. This work led to our central hypothesis: autophagy variation within a tumor cell population controls cell fat in a tumor cell autonomous manner by regulating the efficiency of apoptosis. This mechanism also controls non-cell autonomous signals from dying cells that regulate growth of therapy-resistant tumor subclones. This hypothesis will be tested with the following aims. Specific Aim 1. Determine how autophagy variation in a population of tumor cells controls tumor cell fate to govern response to anti-cancer treatments. Specific Aim 2. Determine how autophagy regulates PUMA to control tumor cell fate after mitochondrial apoptosis. Specific aim 3. Determine role of autophagy's influence on non-cell autonomous signaling in the response of therapy resistant tumor cells in the tumor population. Each aim builds on extensive preliminary data (some not yet published) and proposes complementary approaches that are intended to uncover the central mechanisms that control how autophagy regulates tumor cell fate. We believe these studies will provide new insights into tumor cell life/death decisions and help create a framework for applying these ideas to improve cancer treatment. Importantly, and, we believe, increasing potential overall impact, although this work focuses on what these mechanisms mean for cancer and its treatment, the mechanisms we are studying have important implications for cell fate decisions in other diseases and during normal physiological processes where the interplay between autophagy and apoptosis is also critical.